Differential response of normal and HPV immortalized ectocervical epithelial cells to B[a]P

Cigarette smoking has been established as a risk factor forthe development of cervical cancer. Polycydlic aromatic hydrocarbonssuch as benzo (B[a]P) which are present in cigarette smoke,might account for this increased risk. The effects of B[a]Pon cell growth, aryl hydrocarbon hydroxylase, DNA adducts andp53 levels was measured in cervical cells. Since 90% of cervicalpreneoplastic lesions are positive for the human papillomavirus(HPV) we compared the effects of these chemicals in normal ectocervicalepithelial cells (ECE) and human papillomavirus 16 (HPV16) immortalizedectocervical epithelial cells (ECE16-1). Exposure of normalECE and HPV immortalized ECE16-1 cells to B[a]P inhibited cellproliferation. Inhibition occurred at 20-fold lower concentrationsin the normal ECE cells compared to ECE16-1 cells. The proliferationof cervical cells which express mutated p53 was unaffected byB[a]P. Neither cervical stromal cells nor endometrial stromalcells were affected by these compounds. The effects of B onnormal ECE cell proliferation correlated with increased terminaldifferentiation as measured by increased envelope formation.In contrast, B[a]P exposure did not induce envelope formationin immortalized ECE16-1 cells or in cervical tumor cells. Pretreatmentof both ECE and ECE16-1 cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin,which induces P450 expression and activity, did not alter Bmetabolism in either normal or immortalized cells. Furthermore,equivalent levels of DNA adducts were formed by B[a]P in ECEand ECE16-1 cells. Neither the extent of adduct formation northe rate of their removal differed in normal and Immortalizedcervical cells. There fore, the diminished growth inhibitionof the ECE16-1 cells as compared to normal ECE cells by B[a]is not due to changes in cytochrome P450 of the 1A family metabolismor DNA adduct number. Furthermore, analysis of the p53 levelsin both normal and ECE16-1 cells revealed that p53 levels arehigher in normal versus immortalized ectocervical cells, andp53 is induced in both cell types following B[a]P treatment.Thus reduced p53 levels in ECE16-1 cells may contribute to alack of growth suppression following B[a]P treatment. Theseresults demonstrate that HPV16 immortalization diminishes ectocervicalepithelial cell responsiveness to toxicant damage (i.e. decreasedcell proliferation and increased terminal differentiation).As a result, ECE16-1 cells that sustain genotoxic damage whichleads to DNA adduct formation continue to proliferate and maybe at increased risk for mutations and further progression towardsa fully transformed phenotype.     

DNA adduct level in lung tissue may act as a risk biomarker of lung cancer

Lung cancer is a leading cause of mortality in Taiwan. We hypothesised that high susceptibility to DNA damage in the target organ acts as a risk biomarker for the development of lung cancer. To verify this hypothesis, the aromatic/hydrophobic DNA adduct levels of non-tumorous adjacent lung tissues from 73 primary lung cancer patients and 33 non-cancer controls were evaluated by 32P-postlabelling assay. Wilcoxon rank sum test showed that DNA adduct levels in lung cancer patients (49.58+/-33.39 adducts/10(8) nucleotides) were significantly higher than those in non-cancer controls (18.00+/-15.33 adducts/10(8) nucleotides, P<0.001). The DNA adduct levels among lung cancer and non-cancer samples were not influenced by smoking behaviour and cigarette consumption. Our data also showed that the polymorphisms of cytochrome P4501A1 (CYP1A1) Msp1, glutathione S-transferase M1 (GSTM1) and the combination of both genetic polymorphisms were not related to the DNA adduct levels. Interestingly, positive association between CYP1A1 protein expression and DNA adduct levels was found when CYP1A1 protein expression in lung specimens from lung cancer patients was examined by immunohistochemistry. Multivariate linear regression analysis indicated that the DNA adduct level was not associated with gender, smoking behaviour, or genetic polymorphisms of CYP1A1 and GSTM1. Moreover, multivariate logistic regression analysis showed that persons with high DNA adduct levels (>48.66 adducts/10(8) nucleotides) had an approximately 25-fold risk of lung cancer compared with persons with low DNA adduct levels (相似文献   

Comparative response of normal and of human papillomavirus-16 immortalized human epithelial cervical cells to benzo[a]pyrene.

Laboratory evidence suggests synergism of human papillomavirus (HPV) infection with cigarette smoking behaviors in enhancing the risk of cervical cancer. In this preliminary investigation, we tested the hypothesis that HPV infection may alter the metabolic activation of tobacco smoke carcinogens, such as benzo[a]pyrene (B[a]P), thereby playing a role in the etiology of cervical cancer. We examined in vitro the metabolism and DNA adduct formation of [3H]B[a]P in normal and HPV-16 immortalized human epithelial cervical cells in culture, and investigated the effect of [3H]B[a]P on growth of these cells. Cultures of normal human cervical cells and of HPV-16 immortalized cervical epithelial cells were exposed to 0.2 microM [3H]B[a]P for 24 and 48 h. [3H]B[a]P inhibited growth of both normal and HPV-16 immortalized cervical cells. However, the growth inhibition of normal cells was more profound than that of HPV-16 immortalized cells. Comparison of the metabolism of [3H]B[a]P in these cells indicated that they both metabolize [3H]B[a]P predominantly to [3H]trans-9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene ([3H]B[a]P-9, 10-diol), [3H]r-7,t-8, 9,c-10-tetrahydroxy-7,8,9, 10-tetrahydrobenzo[a]pyrene ([3H]trans-anti-B[a]P-tetraol), and unknown polar products. Enzymatic hydrolysis of water-soluble metabolites indicated that the levels of glucuronide and sulfate conjugates in these cells are negligible. Similarly, both cell lines form similar [3H]B[a]P-DNA adducts. However, the level of the (+)[3H] anti-B[a]P diol epoxide (BPDE)-deoxyguanosine adduct in HPV-16 immortalized cells after 24 and 48 h exposures was 3.8 and 3. 1 pmol/mg DNA, respectively, which is 2.2-fold and 2.6-fold greater than the level of this adduct in normal cells. Under the conditions and within the time frame employed in these assays, both the cell growth and DNA damage induced by [3H]B[a]P appear to be higher in HPV-16 immortalized cells than those detected in normal cells. The results, although preliminary, suggest that HPV-16 immortalized cervical cells are more susceptible to DNA damage by BaP which, in part, may enhance their transformation to malignant cells.     

Polyphenol associated-DNA adducts in lung and blood mononuclear cells from lung cancer patients

The formation of smoking induced-DNA adducts is a critical factor in the induction of human lung cancer. As derivates of benzene and polyaromatic hydrocarbons (PAHs) are important compounds of tobacco smoke, in DNA isolated from human lung and blood mononuclear cells (MNCs) from 38 lung cancer patients, we used the (32)P-postlabeling assay to detect polyphenol associated DNA adducts. Two DNA adducts were detected in blood MNCs and lung tissue that co-chromatographed with DNA modifications from HL60 cells treated with combinations of benzene metabolites (e.g., hydroquinone and benzenetriol). These adducts were designated polyphenol-associated DNA adducts. Relative adduct levels for polyphenolic adducts were five-fold higher than aromatic adducts in both lung and MNCs. A significant correlation was observed between levels of polyphenol adducts and total duration of cigarette smoking in lung (r=0.34; P<0.04) and MNCs (r=0.7; P<0.04), but no correlation between levels of polyphenol adducts and pack-years consumption of cigarettes nor time since quitting smoking in former smokers. Long term former smokers and the one non-smoker in the study had detectable levels of polyphenol adducts. Surprisingly, the levels of polyphenol adducts in MNCs were highly correlated with aromatic adduct levels (r=0.84; P<0.001). Individual aromatic adducts in MNCs also correlated with polyphenol adducts. Total polyphenol adduct levels had a correlation with aromatic DNA adduct levels in lung tissue (r=0.46; P<0.01). To our knowledge these results are the only comparison of adducts in MNCs with lung tissue, and the only data set indicating that blood MNCs are a valid surrogate for lung adduct DNA burden.     

Early age at smoking initiation and tobacco carcinogen DNA damage in the lung

BACKGROUND: DNA adducts formed as a consequence of exposure to tobacco smoke may be involved in carcinogenesis, and their presence may indicate a high risk of lung cancer. To determine whether DNA adducts can be used as a "dosimeter" for cancer risk, we measured the adduct levels in nontumorous lung tissue and blood mononuclear cells from patients with lung cancer, and we collected data from the patients on their history of smoking. METHODS: We used the 32P-postlabeling assay to measure aromatic hydrophobic DNA adducts in nontumorous lung tissue from 143 patients and in blood mononuclear cells from 54 of these patients. From the smoking histories, we identified exposure variables associated with increased DNA adduct levels by use of multivariate analyses with negative binomial regression models. RESULTS/ CONCLUSIONS: We found statistically significant interactions for variables of current and former smoking and for other smoking variables (e.g., pack-years [number of packs smoked per day x years of smoking] or years smoked), indicating that the impact of smoking variables on DNA adduct levels may be different in current and former smokers. Consequently, our analyses indicate that models for current and former smokers should be considered separately. In current smokers, recent smoking intensity (cigarettes smoked per day) was the most important variable. In former smokers, age at smoking initiation was inversely associated with DNA adduct levels. A highly statistically significant correlation (r=.77 [Spearman's correlation]; two sided P<.001) was observed between DNA adduct levels in blood mononuclear cells and lung tissue. IMPLICATIONS: Our results in former smokers suggest that smoking during adolescence may produce physiologic changes that lead to increased DNA adduct persistence or that young smokers may be markedly susceptible to DNA adduct formation and have higher adduct burdens after they quit smoking than those who started smoking later in life.     

Human papillomavirus infection and invasive cervical cancer in Paraguay

HPV types 16 and 18 have been categorized as human carcinogens based on their strong associations with cervical cancer in previous case-control studies. Recent IARC studies in the Philippines, Thailand and Morocco show strong associations between invasive cervical cancer and less common HPV types, including HPV 31, 33, 45, 51, 52 and 58. We present results of a further IARC case-control study conducted in Asunción, Paraguay, to examine the association between specific HPV types and invasive cervical cancer as well as risk factors other than HPV. One-hundred thirteen incident histologically confirmed invasive cervical cancer cases and 91 age-matched hospital controls were recruited. A standardized questionnaire was administered to investigate known and suspected risk factors for cervical cancer. For HPV status determination, cervical biopsy specimens from case subjects and exfoliated cervical cells from control subjects were obtained. HPV DNA was ascertained using a GP5+/6+ PCR-based assay capable of detecting more than 33 HPV types. Overall HPV prevalence was 97% in the cervical cancer cases and 20% in the control subjects. As a single infection, HPV 16 was the predominant type with a prevalence of 48% among case subjects and 5.5% among control subjects. Significant associations with the risk of cervical cancer were detected as follows: any HPV type (OR = 114; 95% CI: 36-361); HPV 16 (OR = 910); HPV 18 (infinite OR); HPV 31 (OR = 110); HPV 33 (OR = 261); HPV 45 (OR = 129); and HPV 58 (OR = 36). In the multivariate model, risk factors other than HPV significantly associated with cervical cancer risk were a higher number of lifetime sexual partners, lower educational status and never having had a Pap smear. Strong associations were found between invasive cervical cancer and specific HPV types 16, 18, 31, 33, 45 and 58.     

DNA adducts in different tissues of smokers and non-smokers

Purified DNA from human lung, liver, bladder, pancreas, breast and cervix has been analysed for DNA adducts using the nuclease P1 modification of the 32P post-labelling technique. Tissues were obtained at autopsy from 13 men and 6 women. Relatives were asked to provide information on smoking history for deceased subjects. All tissues examined except the breast had detectable adducts. In lung, bladder and pancreatic tissue a characteristic pattern of adducts was seen which has previously been reported as typical of cigarette-smoke-induced damage. Smokers and former smokers tended to have higher adduct levels than non-smokers in the tissues examined but this was only significant for the lung. There appeared to be considerable variation in adduct levels among smokers which could not be accounted for by duration or daily consumption level. Certain smokers had high adduct levels in all tissues examined, whilst in others high levels were only seen in some tissues. All cervical samples examined had detectable adducts. These results confirm the finding that cigarette smoking is associated with DNA damage in the lung and suggest that similar damage may be related to tobacco-induced neoplasms of other tissues.     

DNA adducts in human nasal mucosa and white blood cells from smokers and non-smokers

The goal of the present study was to measure the levels of DNA adducts in human nasal mucosa cells and in total white blood cells in relation to smoking. DNA was isolated from samples of 20 healthy volunteers (six smokers and 14 non-smokers). The levels of DNA adducts were measured by 32P-postlabelling assay. In smokers the mean DNA adduct levels were 3.3 and 17.0 adducts/10(8) nucleotides in total white blood cells and nasal mucosa cells respectively. The corresponding values in non-smokers were 2.0 and 6.8 adducts/10(8) nucleotides. The mean adduct level was significantly higher in nasal mucosa cells than in total white blood cells both in smokers and non-smokers. The mean adduct levels in smokers' nasal mucosa cells were significantly higher than those in non- smokers. Thus the nasal mucosa cells constituted a sensitive tissue for the determination of cigarette smoking induced DNA adducts. Combining the sensitivity of the 32P-postlabelling assay with the specificity of the nasal mucosa to the airborne chemical exposures, the DNA adduct analysis from human nasal mucosa cells represents a method of choice in the assessment of exposure to airborne carcinogens.      

Bulky DNA adducts as risk indicator of lung cancer in a Danish case-cohort study

Little is known of the predictive value of the levels of DNA adducts in terms of cancer risk. We examined the association between bulky DNA adducts and risk of lung cancer in a population-based cohort, comprising of 25,717 men and 27,972 women aged 50-64 years at entry. We included 245 cases (137 men and 108 women) with lung cancer and a comparison group of 255 individuals (137 men and 118 women), matched on sex, age and smoking duration. Bulky adducts in white blood cells collected at enrollment and stored at -150 degrees C were analyzed by (32)P-postlabeling method, using the butanol enrichment procedure. The median level of bulky DNA adducts was 0.196 adduct/10(8) nucleotides (5-95 percentiles: 0.094-0.595) among current smokers who were later diagnosed with lung cancer and 0.163 adduct/10(8) nucleotides (5-95 percentiles: 0.091-0.455) among current smokers in the comparison group. The smoking adjusted incidence rate ratios (IRR) for lung cancer in relation to one log unit (natural logarithm) difference in adduct levels were 1.22 (95% CI 0.85-1.74), 1.33 (95% CI 0.89-1.98) and 0.76 (95% CI 0.39-1.47) among all, current and former smokers, respectively. Current smokers with bulky DNA adduct levels above the median had a significant higher lung cancer rate than those with adduct levels below the median (IRR = 1.61; 95% CI 1.04-2.49). The results are compatible with previous studies, suggesting a slightly higher risk of lung cancer with higher levels of adducts among smokers. Our results indicate that bulky DNA adducts may have a weak association with lung cancer risk.     

Polycyclic aromatic hydrocarbon--DNA adducts in white blood cells from lung cancer patients: no correlation with adduct levels in lung.

Smokers of cigarettes are exposed to a number of carcinogens, including polycyclic aromatic hydrocarbons (PAHs), and are at a high risk for lung cancer. PAHs exert their carcinogenic activity after metabolic activation to reactive intermediates that can damage DNA through adduct formation. Measuring DNA adducts in peripheral white blood cells (WBC) could serve as a means of monitoring human exposure to genotoxic agents and subsequently risk assessment. In this study, DNA from WBC obtained from 39 lung cancer patients was examined for PAH-DNA adducts both in an ELISA using a polyclonal antibody against benzo[a]pyrene 7,8-diol-9,10-epoxide (BPDE)-DNA and the 32P-post-labeling technique. The ELISA results showed BPDE-DNA antigenicity in WBC DNA from 12/38 (32%) patients and adduct levels ranged from 1.5 to greater than 150 adducts in 10(8) nucleotides. The autoradiographs of chromatograms of 32P-post-labeled digests of WBC DNA from the 38 patients showed a variety of adduct spots; relative adduct labeling (RAL) values ranged from 0.3 to 407 adducts in 10(8) nucleotides. In 18 of the 38 (47%) persons an adduct spot was detected that co-chromatographed with the major BPDE-DNA adduct (BPDE-dG); RAL values ranged from 0.03 to 382 adducts in 10(8) nucleotides. Correlations were not significant between the adduct levels in WBC and smoking habits, age or sex. From 20 patients of the same group lung tissue was collected at surgery and examined for PAH-DNA adducts by ELISA and 32P-post-labeling assay. No significant correlation was found between DNA adduct levels in blood and lung. This finding stresses the limitations of the use of WBC as a surrogate for adduct levels in the target organ.     

DNA adducts in normal tissue adjacent to breast cancer: a review

To identify possible extrinsic and intrinsic DNA-damaging factors involved in breast cancer etiology, we measured the level of aromatic and lipid peroxidation-related DNA adducts in samples of normal tissue adjacent to breast tumors obtained from 87 breast cancer patients using 32P postlabeling. Twenty-nine cancer-free women who underwent reduction mammoplasty served as controls. Tissue samples from the breast cancer patients contained significantly higher levels of aromatic DNA adducts (mean +/- SEM: 97.4 +/- 23.4 x 109 nucleotides) than did samples obtained from the controls (mean +/- SEM: 23.5 +/- 6.9 x 109 nucleotides). A bulky benzo[a]pyrene (BP)-like adduct was detected in 41% of the cancer patients, but in none of the controls. The level of this adduct was extremely high in some patients (> 1/106). While 88% of the patients with a smoking history had smoking-specific DNA adducts in their breast tissues, the presence of BP-like adduct was not related to smoking history. The cancer patients also had a significantly higher level of lipid peroxidation-related DNA adducts than did controls. The level of these adducts correlated with the presence of the BP-like adduct. To further explore the origin of the BP-like adduct, we examined the level of polycyclic aromatic hydrocarbon (PAH)-DNA adducts and 8-hydroxyguanine (8-OH-G) in tissue sections obtained from 37 breast cancer patients using immunocytochemistry. We found that patients who had the BP-like adduct showed significantly greater immunostaining for PAH adducts than did those without the BP-like adduct (p = 0.04). In addition, we found that adipocytes tended to have greater immunostaining for the PAH adducts than did epithelial cells. On the other hand, epithelial cells tended to have a higher frequency and greater intensity of staining for 8-OH-G than did adipocytes. The detection of PAH adducts, lipid peroxidation-related DNA adducts, and 8-OH-G in normal breast tissues of breast cancer patients suggests that both exogenous and endogenous DNA-damaging factors may be involved in breast cancer. The exogenous source may involve the types of carcinogen exposure other than cigarette smoking, and the endogenous source may involve oxidative stress associated with normal metabolic activities.     

Covalent DNA damage in tissues of cigarette smokers as determined by 32P-postlabeling assay

Covalent DNA addition products (adducts) formed by the reaction of chemical carcinogens or their metabolites with DNA are critically involved in the initiation of chemical carcinogenesis and may serve as molecular markers and dosimeters for environmental carcinogen exposures. Using a highly sensitive 32P-postlabeling assay for DNA adduct analysis, we studied DNA damage elicited by cigarette smoke in tissues of smokers. A multitude of characteristic smoking-induced, presumably aromatic DNA adducts were found to occur in a dose- and time-dependent manner in the lung, bronchus, and larynx of smokers with cancer of these organs and to decline only slowly after cessation of smoking. Low levels of adducts appeared to persist for up to 14 years in the lungs of exsmokers with high previous exposures. These results corroborate data of epidemiological studies showing that the lung cancer risk and mortality of smokers increase with the intensity and duration of smoking and decline only slowly after cessation of smoking. Tissue distribution studies in autopsy samples revealed the presence of smoking-associated DNA lesions also in the kidney, bladder, esophagus, heart, ascending aorta, and liver. The most extensive DNA damage was found in lung and heart, i.e., 1 aromatic adduct in about 10(7) DNA nucleotides. Our results suggest that cigarette smoking-induced DNA adduct formation is causally related to cancer in the target organs.     

Prevalence and distribution of human papillomavirus type-16 DNA in pelvic lymph nodes of patients with cervical cancer and in women with no history of cervical abnormality.

The polymerase chain reaction (PCR) was used to investigate the prevalence and distribution of human papillomavirus (HPV)-16 DNA in paraffin sections of all pelvic lymph nodes removed from 14 patients with Stage Ib-cervical cancer at the time of resection of their primary tumours. The results were compared with those obtained from 8 women with no known history of cervical abnormality. In all, 22 cervical biopsies and 40 I lymph nodes (296 paraffin blocks) were examined. Nine of the 14 cervical cancer patients had primary tumours that were positive for HPV-16 DNA: only 3 of these had lymph nodes with histological evidence of metastasis, and HPV 16 DNA was detected in each of the corresponding paraffin blocks. HPV 16 DNA was also detected in varying proportions (8%-92%) of the histologically-negative lymph nodes from these women. There was no correlation between the HPV DNA-positive lymph nodes and their proximity to the primary tumour. HPV-16 DNA was not identified in any of the lymph nodes from the 5 women whose cancers were not HPV-16-related, or in those of women with no evidence of cervical abnormality. This preliminary survey suggests that HPV DNA is frequently transported from HPV-16-related cervical tumours to regional lymph nodes. However, its practical significance will not be clear until sufficient time has elapsed for correlation of the results with the clinical outcome.     

子宫颈癌患者人乳头瘤病毒16、18检测

 目的　探讨人乳头瘤病毒（HPV）16、18型在子宫颈癌的发生、发展中的意义。方法　应用实时荧光定量聚合酶链反应技术对子宫颈原位癌13例，子宫颈癌Ⅰ期32例，子宫颈上皮内瘤样病变（CIN）Ⅰ~Ⅱ级12例，对照组54例（慢性子宫颈炎组37例、非研究疾病组17例）进行HPV16、HPV18型荧光基因定量检测，计算出HPV DNA的拷贝数。结果　研究组、对照组HPV16、HPV18的感染率差异有统计学意义（P＜0.05），在子宫颈癌发生的不同阶段，HPV16、HPV18的感染定量差异亦有统计学意义。子宫颈癌HPV含量与肿瘤直径大小、浸润间质深度、淋巴结阳性个数无相关性（r = 0.168， r = 0.280， r = 0.333，P＞0.05）；局部肿瘤的直径大小与浸润间质深度呈正相关（r = 0.473，P＜0.05）。结论　致癌性HPV的持续、高浓度存在是子宫颈癌发生、发展的主要因素之一。     

Human papillomavirus 16E6 oncogene mutation in cervical cancer

Objective: Cervical cancer (CC) is the second most common type of cancer in women worldwide, after breast cancer. High-risk human papillomaviruses (HR-HPVs) are considered to be the major causes of cervical cancer. HPV16 is the most common type of HR-HPVs and HPV16 E6 gene is one of the major oncogenes. Specific mutations are considered as dangerous factors causing CC. This study was designed to find mutations of HPV16 E6 and the relationship between the mutations and the happening of CC.Methods: The tissue DNA was extracted from 15 biopsies of CC. Part of HPV16 E6 gene (nucleotide 201-523) was amplified by polymerase chain reaction (PCR) from the CC tissue DNA. The PCR fragments were sequenced and analyzed.Results: The result of PCR showed that the positive rate of HPV16 E6 was 93.33% (14/15). After sequencing and analyzing, in the 13 out of 14 PCR fragments, 4 maintained prototype (30.77%), 8 had a same 350G mutation (61.54%), and 1 had a 249G mutation (7.69%).Conclusion: This study suggest that there is a high infection rate of HPV in cervical cancer and most of the HPV16 E6 gene has mutations. Those mutations may have an association with the development of cervical cancer.     

Antibodies against high‐risk human papillomavirus proteins as markers for invasive cervical cancer

Different human papillomavirus (HPV) genes are expressed during the various phases of the HPV life cycle and may elicit immune responses in the process towards malignancy. To evaluate their association with cervical cancer, antibodies against proteins from HPV16 (L1, E1, E2, E4, E6 and E7) and HPV18/31/33/35/45/52/58 (L1, E6 and E7) were measured in serum of 307 invasive cervical cancer cases and 327 controls from Algeria and India. Antibody response was evaluated using a glutathione S‐transferase‐based multiplex serology assay and HPV DNA detected from exfoliated cervical cells using a GP5+/6+‐mediated PCR assay. Among HPV16 DNA‐positive cases, seroprevalence of HPV16 antibodies ranged from 16% for HPV16 E1 to 50% for HPV16 E6 and all were significantly higher than controls. Seroprevalence of E6, E7 and L1 antibodies for HPV18 and for at least one of HPV31/33/35/45/52/58 were also higher in cases positive for DNA of the corresponding type (50% and 30% for E6 of HPV18 and HPV31/33/35/45/52/58 combined, respectively). E6 and E7 antibodies were rarely found in controls, but cross‐reactivity was evident among cancer cases positive for DNA of closely phylogenetically‐related HPV types. E6 or E7 antibodies against any of the eight HPV types were detected in 66.1% of all cervical cancer cases, as compared to 10.1% of controls. E6, and to a lesser extent E7, antibodies appear to be specific markers of HPV‐related malignancy. However, even among cases positive for the same type of HPV DNA, approximately one‐third of cervical cancer cases show no detectable immune response to either E6 or E7.     

Formation of mitoxantrone adducts in human tumor cells: potentiation by AN-9 and DNA methylation

The ability of mitoxantrone to form DNA adducts was investigated in a series of human tumor cell lines consisting of human cervical cancer (HeLa), human breast cancer (MCF-7), and human neuroblastoma (IMR-32) cells. The mitoxantrone-resistant human promyelocytic leukemia cell line HL60/MX2 was also compared to the parental cell line HL60 in terms of adduct formation in cellular DNA, RNA, and protein. DNA adduct formation detected using [14C]mitoxantrone as a single agent occurred at very low levels but addition of the formaldehyde-releasing prodrug AN-9 (pivaloyloxymethyl butyrate) increased adduct formation considerably in all cell lines tested. Adduct formation increased when increasing ratios of AN-9 were used, and were observed at maximal levels when AN-9 addition was 4 h after the addition of mitoxantrone. However, low levels of adducts were observed when AN-9 addition was 16 h prior to mitoxantrone. The ability of [14C]mitoxantrone to form adducts with DNA, RNA, and protein was assessed in HL60 cells, and DNA was found to be the major substrate for adduct formation. RNA was also shown to be a good substrate while protein adduct levels were consistently very low. In mitoxantrone-resistant HL60/MX2 cells, DNA adduct levels were approximately fourfold lower. To establish the influence of DNA methylation on the ability of mitoxantrone to form adducts in cells, decitabine was used to reduce DNA methylation levels in cells prior to mitoxantrone treatment. This was clearly shown to influence adduct formation, with increasing decitabine levels leading to a decrease in the level of adducts observed in both IMR-32 and MCF-7 cell lines. Collectively, these results suggest that two major factors that influence the extent of mitoxantrone adduct formation in cells are the availability of formaldehyde and the extent of genomic DNA methylation.     

Sensitivity to DNA damage induced by benzo(a)pyrene diol epoxide and risk of lung cancer: a case-control analysis

Levels of DNA adducts vary greatly in vivo, attributable to individual differences in enzymatic bioactivation of benzo(a)pyrene. We developed an assay to measure the levels of DNA adducts induced in vitro by benzo(a)pyrene diol epoxide (BPDE), a bioactivated form of benzo(a)pyrene. In this large molecular epidemiological study of lung cancer, we tested the hypothesis that the level of in vitro BPDE-induced adducts is associated with risk of lung cancer. This hospital-based case-control study included 221 newly diagnosed lung cancer cases and 229 healthy controls frequency matched on age, sex, ethnicity, and smoking status. Short-term cultured peripheral blood lymphocytes from each subject were exposed in vitro to BPDE (4 microm) for 5 h, and the 32P-postlabeling method was then used to measure BPDE-induced DNA adducts in the host cells. Overall, the patients had significantly higher levels of BPDE-DNA adducts than did the controls (mean +/- SD per 107 nucleotides, 93.2+/-89.3 for cases versus 63.7+/-61.1 for controls; P = 0.001). Univariate and multivariate logistic regression analyses were performed to calculate the crude and adjusted odds ratios and their 95% confidence intervals. When the median adduct level of controls (46/10(7) nucleotides) was used as the cutoff point, 64% of cases had higher levels (odds ratio, 2.15; 95% confidence interval, 1.39-3.33, adjusted for age, sex, ethnicity, body mass index, recent weight loss, pack-years smoked, smoking in the last 24 h, and family history of cancer). Stratified analyses showed consistently higher levels of BPDE-induced adducts in cases than in controls, regardless of subgroup of age, sex, ethnicity, body mass index, recent weight loss, pack-years smoked, smoking in the last 24 h, and family history of cancer. A significant dose-response relationship between the quartile levels of BPDE-induced DNA adducts and the risk of lung cancer was observed (trend test, P < 0.001). The significant association between the level of in vitro BPDE-induced DNA adducts and risk for lung cancer suggests that subjects very sensitive to BPDE-induced DNA damage may have a suboptimal ability to remove the BPDE-DNA adducts and so are susceptible to tobacco carcinogen exposure and, therefore, may be at increased risk of lung cancer.     

DNA adducts in bronchial biopsies

To investigate the feasibility of measuring DNA-carcinogen adducts in the lungs of non-surgical patients, endobronchial biopsies were obtained from 78 patients undergoing routine diagnostic bronchoscopy. Lung cancer was present in 37 (47%) of the patients. DNA was isolated from the tissues and analyzed by HPLC- or nuclease-PI-enriched 32P-postlabelling, using procedures selective for aromatic adducts. Chromatograms from all 28 current smokers showed a distinctive diagonal adduct zone which was present in only 24 of 40 ex-smokers and 4 of 10 lifetime non-smokers. Adduct levels and chromatographic patterns were similar in bronchial tissue from different lobes of the lung, in bronchial and alveolar tissue, and in tumor and non-tumor bronchial tissue taken from the same subject. Bronchial DNA adduct levels were strongly associated with cigarette smoking status and dropped rapidly after smoking ceased. Higher levels of DNA adducts seen in the lung-cancer patients were mainly due to cigarette smoking. Frequent alcohol intake was the only dietary factor associated with higher levels of bronchial DNA adducts. We conclude that the level of bronchial DNA adducts is strongly associated with cigarette-smoking history and with alcohol intake, but is not associated with lung cancer independently from its relation to smoking. The results indicate the feasibility of using 32P-postlabelling to detect and quantitate genetic damage in bronchial biopsy specimens.     

Immunoglobulin-A and -G responses against virus-like particles (VLP) of human papillomavirus type 16 in women with cervical cancer and cervical intra-epithelial lesions

Immunoglobulin-A and -G (IgA and IgG) responses against HPV-16-like particles (VLP) were tested by ELISA in 104 women with cervical abnormalities, 26 atypical cells of undetermined significance (ASCUS) and 14 cytologically normal women with HPV DNA. As controls, 130 age-matched cytologically normal women with no HPV DNA were selected from the population in which the cases were generated. The existence of HPV DNA in cervical samples was tested by a PCR-based method. The normal women positive with HPV16 DNA were followed up at 4- to 7-month intervals for 16 to 24 months. IgA and IgG antibodies against HPV-16 VLP were frequently detected in these women repeatedly positive with HPV-16 DNA, suggesting that persistent HPV infection is crucial for effective antibody responses against the viruses. IgA response appears earlier and persists longer than IgG response. Women with HPV DNA of types 16, 31/33/35, 58 and unknown types showed significantly higher seropositivity for both IgA and IgG antibodies than the controls (p < 0.05 for both). No significant seropositivity for IgA or IgG was detected in the HPV-18/45-DNA-positive group. HPV 31/33/35, 58 appear to be types close to HPV 16, whereas HPV 18/45 appears to be distinct from HPV 16 in antigenicity. IgA and IgG responses against HPV-16 VLP were more frequently observed in women with normal cervices with HPV DNA, ASCUS, HSIL and cervical cancer than in the controls. Strong IgA and IgG responses depended on HPV-16 infection in HSIL and cervical cancer, but there was no correlation between the serological responses and the status of HPV DNA in ASCUS and LSIL. Antibody positivity reflects persistent viral infection that may increase the risk for malignant progression of the cervix. This serological assay using HPV 16-VLP may therefore be useful as a new diagnostic tool supplementing cervical cytological tests. Int. J. Cancer 75:529–535, 1998.© 1998 Wiley-Liss, Inc.